Mutant EGIII cellulase, DNA encoding such EGIII compositions and methods for obtaining same

ABSTRACT

The present invention relates to variant EGIII cellulases that have improved stability and/or performance. The variant cellulases have replacements at sensitive residues to improve stability and/or performance.

This application is a divisional of Ser. No. 10/075872, filed Feb. 13,2002, now U.S. Pat. No. 6,500,211, which is a continuation of Ser. No.09/633084, filed Aug. 4, 2000, now U.S. Pat. No. 6,407,046, which is acontinuation-in-part of Ser. No. 09/146770, filed Sep. 3, 1998, now U.S.Pat. No. 6,187,732.

This application is also related to concurrently filed applications withare U.S. Ser. Nos. 09/632,575, 09/632,426, 09/632,570, and 09/633,085,filed on Aug. 4, 2000, and all of which are incorporated by reference intheir entirety.

GOVERNMENT SPONSORED RESEARCH AND DEVELOPMENT

Not Applicable.

BACKGROUND OF THE INVENTION

Cellulases are enzymes that are capable of hydrolysis of theβ-D-glucosidic linkages in celluloses. Cellulolytic enzymes have beentraditionally divided into three major classes: endoglucanases,exoglucanases or cellobiohydrolases and β-glucosidases (Knowles, J. etal., (1987), TIBTECH 5, 255-261); and are known to be produced by alarge number of bacteria, yeasts and fungi.

Although cellulases are used to degrade wood pulp and animal feed,cellulases are primarily used in the treatment of textiles, e.g., indetergent compositions for assisting in the removal of dirt or grayishcast (see e.g., Great Britain Application Nos. 2,075,028, 2,095,275 and2,094,826) or in the treatment of textiles prior to sale to improve thefeel and appearance of the textile. Thus, Great Britain Application No.1,358,599 illustrates the use of cellulase in detergents to reduce theharshness of cotton containing fabrics.

Cellulases have also been used in the treatment of textiles torecondition used fabrics by making their colors more vibrant (see e.g.,The Shizuoka Prefectural Hammamatsu Textile Industrial ResearchInstitute Report 24:54-61 (1986)). Repeated washing of cotton containingfabrics results in a grayish cast to the fabric which is believed to bedue to disrupted and disordered fibrils, sometimes called “pills”,caused by mechanical action. This greyish cast is particularlynoticeable on colored fabrics. As a consequence, the ability ofcellulase to remove the disordered top layer of the fiber and thusimprove the overall appearance of the fabric has been of value.

Because of its effectiveness in many industrial processes, there hasbeen a trend in the field to search for specific cellulase compositionsor components that have particularly effective performance profiles withrespect to one or more specific applications. As possible sources ofcellulases, practitioners have focused on fungi and bacteria. Forexample, cellulase produced by certain fungi such as Trichoderma spp.(especially Trichoderma reesei) have been given much attention because acomplete cellulase system capable of degrading crystalline forms ofcellulose is readily produced in large quantities via fermentationprocedures. This specific cellulase complex has been extensivelyanalyzed to determine the nature of its specific components and theability of those components to perform in industrial processes (see,Wood et al., “Methods in Enzymology”, 160, 25, pages 234, et seq.(1988). U.S. Pat. No. 5,475,101 (Ward et al.) discloses the purificationand molecular cloning of one particularly useful enzyme calledendoglucanase III (EGIII) which is derived from Trichoderma reesei.

PCT Publication No. WO 94/14953 discloses endoglucanases that areencoded by a nucleic acid that comprises any one of a series of DNAsequences, each having 20 nucleotides.

Ooi, et al., Curr. Genet. 18:217-222 (1990) disclose the cDNA sequencecoding for endoglucanase F1-CMC produced by Aspergillus aculeatus thatcontains the amino acid strings NNLWG, ELMIW and GTEPFT. Sakamoto, etal., Curr. Genet. 27:435-439 (1995) discloses the cDNA sequence encodingthe endoglucanase CMCase-1 From Aspergillus kawachii IFO 4308 whichcontains the amino acid strings ELMIW and GTEPFT. Ward, et al.,discloses the sequence of EGIII having the amino acid strings NNLWG,ELMIW and GTEPFT. Additionally, two cellulase sequences, one fromErwinia carotovara and Rhodothermus marinus are disclosed in Saarilahti,et al., Gene 90:9-14 (1990) and Hreggvidsson, et al., Appl. Environ.Microb. 62:3047-3049 (1996) that contain the amino acid string ELMIW.

Despite knowledge in the art related to many cellulase compositionshaving applications in some or all of the above areas, there is acontinued need for new cellulase compositions which have improvedstability under conditions present in applications for which cellulasesare useful, e.g., household and laundry detergents and textile treatmentcompositions.

SUMMARY OF THE INVENTION

A variant EGIII cellulase is provided wherein the variant comprises asubstitution at a residue that is sensitive to surfactant and/ortemperature stress and is derived from T. reesei EGIII cellulase. In apreferred embodiment, the variant comprises a substitution or deletionat a position corresponding to one or more of residues W7, T11, T16,A35, S39, G41, S63, A66, S77, N91, S143, T163, N167 and/or, A188. In amore preferred embodiment, the variant comprises a substitution at aposition corresponding to one or more of residues W7Y, T11S, T16I, A35S,S39N, G41A, S63V, A66N, S77G, N91D, S143T, T163S, N167S and/or, A188G.

In another embodiment of the invention, a DNA encoding the variant EGIIIcellulase is provided. In a preferred aspect of this embodiment, the DNAis in a vector. In another aspect of this embodiment, the vector is usedto transform a host cell.

In yet another embodiment, a method of producing a variant EGIIIcellulase having improved stability and/or performance is provided. Themethod comprises the steps of culturing a host cell in a suitableculture medium under suitable conditions to produce cellulase andobtaining the produced cellulase. In another embodiment a detergentcomposition comprising a surfactant and a variant EGIII cellulase isprovided, wherein the variant EGIII cellulase comprises a substitutionat residue sensitive to surfactant and/or temperature. In a preferredembodiment, the variant comprises a substitution or deletion at aposition corresponding to one or more of residues W7, T11, T16, A35,S39, G41, S63, A66, S77, N91, S 143, T163, N167 and/or, A188. In a morepreferred embodiment, the variant EGIII cellulase comprises asubstitution at a position corresponding to one or more of residues W7Y,T11S, T16I, A35S, S39N, G41A, S63V, A66N, S77G, N91D, S143T, T163S,N167S and/or, A188G. In another aspect of this embodiment, the detergentis a laundry or a dish detergent.

In another embodiment of this invention, the variant EGIII cellulase isused in the treatment of a cellulose-containing textile, preferably tostone wash indigo dyed denim. In another embodiment, the variant is usedas a feed additive. In yet another embodiment, the variant is used inthe treatment of wood pulp. In still another embodiment, the variant isused in the reduction of biomass to glucose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the amino acid sequence of EGIII from Trichodermareesei.

FIG. 2 illustrates the DNA sequence of EGIII from Trichoderma reeseiwithout introns.

FIG. 3 is a schematic showing the alignment of amino acids in EGIII andEGIII-like cellulases.

DETAILED DESCRIPTION OF THE INVENTION

Applicants have isolated a novel cellulase from Hypocrea schweinitziithat has significant homology to EGIII from Trichoderma reesei. Analysisof this cellulase has resulted in the discovery that substantialdifferences exist in terms of performance between the two cellulases,despite the significant homology. In fact, the homologous enzyme hassignificantly diminished performance under conditions of thermal stressor in the presence of surfactants. This discovery is particularlyinteresting as EGIII differs from its Hypocrea schweinitzii homolog inonly 14 positions indicating that these 14 positions lie in portions orareas of the protein that have a significant impact on the stabilityand/or performance of EGIII. Thus, Applicants discovered that byoptimizing residues in EGIII at one or more of the 14 differentpositions or spatially near them, it is possible to optimize theperformance of EGIII.

Accordingly, the present invention relates to a variant EGIII cellulasehaving improved performance in the presence of, e.g., surfactant and/orthermal mediated stress. The variant is characterized by replacement ofone or more residues identified herein as being critical for stabilityand/or performance with a residue that confers improved stability and/orperformance to the enzyme. Preferably, but not necessarily, thesensitive residue is replaced with a residue that has improvedoxidative, alkaline or thermal stability compared to the wild type (T.reesei EGIII) residue at that position. Suitable substitutions may beany substitution that modifies stability, particularly preferredsubstitutions being those which provide improved stability and mostpreferred substitutions being those which provide conservativemodifications in terms of charge, polarity and/or size. As anon-limiting example, substitutions which are particularly of valueinclude substitutions wherein leucine is modified to an isoleucine,isoleucine is modified to a leucine, tryptophan is modified to atyrosine, threonine is modified to an asparagine, alanine is modified toa glycine, serine is modified to an asparagine, glycine is modified to aproline and asparagine is modified to a threonine.

Definitions

Within the specification, certain terms are disclosed which are definedbelow so as to clarify the nature of the claimed invention.

“Cellulase” is a well-classified category of enzymes in the art andincludes enzymes capable of hydrolyzing cellulose polymers to shortercello-oligosaccharide oligomers, cellobiose and/or glucose. Commonexamples of cellulase enzymes include exo-cellobiohydrolases andendoglucanases and are obtainable from many species of cellulolyticorganisms, particularly including fungi and bacteria.

“EGIII cellulase” refers to the endoglucanase component described inU.S. Pat. No. 5,475,101 and Proceedings on the Second TRICEL Symposiumon Trichoderma Reesei Cellulases And Other Hydrolases, Suominen &Reinikainen eds., Espoo Finland (1993), pp. 153-158 (Foundation forBiotechnical and Industrial Fermentation Research, Vol. 8). As discussedtherein, EGIII is derived from Trichoderma reesei (reesei) and ischaracterized by a pH optimum of about 5.8, an isoelectric point (pI) ofabout 7.4 and a molecular weight of about 25 kD. The enzyme commonlyreferred to as EGIII from Trichoderma reesei has been previouslyreferred to in the literature by the nomenclature EGIII by some authors,but that enzyme differs substantially from the enzyme defined herein asEGIII in terms of molecular weight, pI and pH optimum.

“Cellulose containing fabric” means any sewn or unsewn fabrics, yarns orfibers made of cotton or non-cotton containing cellulose or cotton ornon-cotton containing cellulose blends including natural cellulosics andmanmade cellulosics (such as jute, flax, ramie, rayon, and lyocell).Included under the heading of manmade cellulose containing fabrics areregenerated fabrics that are well known in the art such as rayon. Othermanmade cellulose containing fabrics include chemically modifiedcellulose fibers (e.g, cellulose derivatized by acetate) andsolvent-spun cellulose fibers (e.g., lyocell). Specifically includedwithin the definition of cellulose containing fabric is any yarn orfiber made of such materials. Cellulose containing materials are oftenincorporated into blends with materials such as synthetic fibers andnatural non-cellulosic fibers such as wool and silk.

A residue in an EGIII homolog from H. schweinitzii which is“corresponding” or “equivalent” to a residue present in EGIII means aresidue which exists in an equivalent position to that in EGIII, asindicated by primary sequence homology, tertiary structural homology (asshown by, e.g., crystal structure or computer modeling) or functionalequivalence.

“Equivalent residues” may also be defined by determining homology at thelevel of tertiary structure for a precursor protease whose tertiarystructure has been determined by x-ray crystallography. Equivalentresidues are defined as those for which the atomic coordinates of two ormore of the main chain atoms of a particular amino acid residue of aEGIII homolog from H. schweinitzii and T. reesei EGIII (N on N, CA onCA, C on C and O on O) are within 0.13 nm and preferably 0.1 nm afteralignment. Alignment is achieved after the best model has been orientedand positioned to give the maximum overlap of atomic coordinates ofnon-hydrogen protein atoms of the EGIII homolog in question to the T.reesei EGIII. The best model is the crystallographic model giving thelowest R factor for experimental diffraction data at the highestresolution available.${R\quad {factor}} = \frac{{\sum\limits_{h}\quad {{{Fo}(h)}}} - {{{Fc}(h)}}}{\sum\limits_{h}\quad {{{Fo}(h)}}}$

Equivalent residues which are functionally analogous to a specificresidue of T. reesei EGIII are defined as those amino acids of acellulase which may adopt a conformation such that they either alter,modify or contribute to protein structure, substrate binding orcatalysis in a manner defined and attributed to a specific residue ofthe T. reesei EGIII. Further, they are those residues of the cellulase(for which a tertiary structure has been obtained by x-raycrystallography) which occupy an analogous position to the extent that,although the main chain atoms of the given residue may not satisfy thecriteria of equivalence on the basis of occupying a homologous position,the atomic coordinates of at least two of the side chain atoms of theresidue lie with 0.13 nm of the corresponding side chain atoms of T.reesei EGIII. The crystal structure of T. reesei EGIII is presented TheProtein Society, Fourteenth Symposium. San Diego, Calif. Aug. 5-9, 2000,the disclosure of which is incorporated by reference in its entirety.The coordinates of CelB of Streptomyces lividais, a homologous member ofthe Family 12 glycosyl hydrolases is provided in Sulzenbacher, et al.,Biochemistry 36:6032 (1997) and in Sulzenbacher, et al., Biochemistry38:4826 (1999).

“Cotton-containing fabric” means sewn or unsewn fabrics, yams or fibersmade of pure cotton or cotton blends including cotton woven fabrics,cotton knits, cotton denims, cotton yarns, raw cotton and the like. Whencotton blends are employed, the amount of cotton in the fabric ispreferably at least about 35 percent by weight cotton. When employed asblends, the companion material employed in the fabric can include one ormore non-cotton fibers including cellulosic or synthetic fibers such aspolyamide fibers (for example, nylon 6 and nylon 66), acrylic fibers(for example, polyacrylonitrile fibers), and polyester fibers (forexample, polyethylene terephthalate), polyvinyl alcohol fibers (forexample, Vinylon), polyvinyl chloride fibers, polyvinylidene chloridefibers, polyurethane fibers, polyurea fibers and aramid fibers.

“Detergent composition” means a mixture that is intended for use in awash medium for the laundering of soiled cellulose containing fabrics.In the context of the present invention, such compositions may include,in addition to cellulases and surfactants, additional hydrolyticenzymes, builders, bleaching agents, bleach activators, bluing agentsand fluorescent dyes, caking inhibitors, masking agents, cellulaseactivators, antioxidants, and solubilizers. Such compositions aregenerally used for cleaning soiled garments and are not used during themanufacturing process, in contrast to stonewashing compositions.Detergent compositions comprising cellulase are described in, forexample, U.S. Pat. No. 5,290,474 and EP Publication No. 271 004,incorporated herein by reference.

“DNA vector” means a nucleotide sequence that comprises one or more DNAfragments or DNA variant fragments encoding an EGIII or variantsdescribed above, which can be used, upon transformation into anappropriate host cell, to cause expression of the EGIII.

“Expression vector” means a DNA construct comprising a DNA sequence thatwhich is operably linked to a suitable control sequence capable ofeffecting the expression of the DNA in a suitable host. Such controlsequences may include a promoter to effect transcription, an optionaloperator sequence to control transcription, a sequence encoding suitableribosome-binding sites on the mRNA, and sequences that controltermination of transcription and translation. Different cell types arepreferably used with different expression vectors. A preferred promoterfor vectors used in Bacillus subtilis is the AprE promoter; a preferredpromoter used in E. coli is the Lac promoter, a preferred promoter usedin Saccharomyces cerevisiae is PGK1, a preferred promoter used inAspergillus niger is glaa, and a preferred promoter for Trichodermareesei (reesei) is cbhI. The vector may be a plasmid, a phage particle,or simply a potential genomic insert. Once transformed into a suitablehost, the vector may replicate and function independently of the hostgenome, or may, under suitable conditions, integrate into the genomeitself. In the present specification, plasmid and vector are sometimesused interchangeably. However, the invention is intended to includeother forms of expression vectors that serve equivalent functions andwhich are, or become, known in the art. Thus, a wide variety ofhost/expression vector combinations may be employed in expressing theDNA sequences of this invention. Useful expression vectors, for example,may consist of segments of chromosomal, non-chromosomal and syntheticDNA sequences such as various known derivatives of SV40 and knownbacterial plasmids, e.g., plasmids from E. coli including col E1, pCR1,pBR322, pMb9, pUC 19 and their derivatives, wider host range plasmids,e.g., RP4, phage DNAs e.g., the numerous derivatives of phage λ, e.g.,NM989, and other DNA phages, e.g., M13 and filamentous single strandedDNA phages, yeast plasmids such as the 2μ plasmid or derivativesthereof, vectors useful in eukaryotic cells, such as vectors useful inanimal cells and vectors derived from combinations of plasmids and phageDNAs, such as plasmids which have been modified to employ phage DNA orother expression control sequences. Expression techniques using theexpression vectors of the present invention are known in the art and aredescribed generally in, for example, Sambrook, et al., MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press(1989). Often, such expression vectors including the DNA sequences ofthe invention are transformed into a unicellular host by directinsertion into the genome of a particular species through an integrationevent (see e.g., Bennett & Lasure, More Gene Manipulations in Fungi,Academic Press, San Diego, pp. 70-76 (1991) and articles cited thereindescribing targeted genomic insertion in fungal hosts, incorporatedherein by reference).

“Functionally attached to” means that a regulatory region, such as apromoter, terminator, secretion signal or enhancer region is attached toa structural gene and controls the expression of that gene.

“Host strain” or “host cell” means a suitable host for an expressionvector comprising DNA according to the present invention. Host cellsuseful in the present invention are generally procaryotic or eucaryotichosts, including any transformable microorganism in which expression canbe achieved. Specifically, host strains may be Bacillus subtilis,Escherichia coli, Trichoderma reesei (reesei), Saccharomyces cerevisiaeor Aspergillus niger Host cells are transformed or transfected withvectors constructed using recombinant DNA techniques. Such transformedhost cells are capable of both replicating vectors encoding swolleninand its variants (mutants) or expressing the desired peptide product. Ina preferred embodiment according to the present invention, “host cell”means both the cells and protoplasts created from the cells ofTrichoderma sp.

“Stonewashing” means the treatment of cellulose containing fabric with acellulase solution under agitating and cascading conditions, e.g., in arotary drum washing machine, to impart a “stonewashed” appearance to thedenim. The cellulase solution according to the instant invention willfunctionally replace the use of stones in such art-recognized methods,either completely or partially. Methods for imparting a stonewashedappearance to denim are described in U.S. Pat. No. 4,832,864, which isincorporated herein by reference in its entirety. Generally,stonewashing techniques have been applied to indigo dyed cotton denim.

“Stonewashing composition” means a formulation for use in stonewashingcellulose containing fabrics. Stonewashing compositions are used tomodify cellulose-containing fabrics prior to presentation for consumersale, i.e., during the manufacturing process. In contrast, detergentcompositions are intended for the cleaning of soiled garments.

“Signal sequence” means a sequence of amino acids bound to theN-terminal portion of a protein that facilitates the secretion of themature form of the protein outside of the cell. This definition of asignal sequence is a functional one. The mature form of theextracellular protein lacks the signal sequence that is cleaved offduring the secretion process.

“Surfactant” means any compound generally recognized in the art ashaving surface-active qualities. Thus, for example, surfactants compriseanionic, cationic and nonionic surfactants such as those commonly foundin detergents. Cationic surfactants and long-chain fatty acid saltsinclude saturated or unsaturated fatty acid salts, alkyl or alkenylether carboxylic acid salts, α-sulfofatty acid salts or esters, aminoacid-type surfactants, phosphate ester surfactants, quaternary ammoniumsalts including those having 3 to 4 alkyl substituents and up to 1phenyl substituted alkyl substituents. Examples of cationic surfactantsand long-chain fatty acid salts are disclosed in British PatentApplication No. 2 094 826 A, the disclosure of which is incorporatedherein by reference. The composition may contain from about 1 to about20 weight percent of such cationic surfactants and long-chain fatty acidsalts.

Anionic surfactants include linear or branched alkylbenzenesulfonates;alkyl or alkenyl ether sulfates having linear or branched alkyl groupsor alkenyl groups; alkyl or alkenyl sulfates; olefinsulfonates; andalkanesul-fonates. Suitable counter ions for anionic surfactants includealkali metal ions such as sodium and potassium; alkaline earth metalions such as calcium and magnesium; ammonium ion; and alkanolamineshaving 1 to 3 alkanol groups of carbon number 2 or 3. Ampholyticsurfactants include quaternary ammonium salt sulfonates, andbetaine-type ampholytic surfactants. Such ampholytic surfactants haveboth the positive and negative charged groups in the same molecule.Nonionic surfactants may comprise polyoxyalkylene ethers, as well ashigher fatty acid alkanolamides or alkylene oxide adduct thereof, fattyacid glycerine monoesters, and the like. Examples of surfactants for usein this invention are disclosed in British Patent Application No. 2 094826 A, the disclosure of which is incorporated herein by reference.Mixtures of such surfactants can also be used.

“Variant” means a protein which is derived from a precursor protein(e.g., the native protein) by addition of one or more amino acids toeither or both the C- and N-terminal end, substitution of one or moreamino acids at one or a number of different sites in the amino acidsequence, deletion of one or more amino acids at either or both ends ofthe protein or, at one or more sites in the amino acid sequence, orinsertion of one or more amino acids at one or more sites in the aminoacid sequence. The preparation of an enzyme variant is preferablyachieved by modifying a DNA sequence which encodes for the nativeprotein, transformation of that DNA sequence into a suitable host, andexpression of the modified DNA sequence to form the variant enzyme. Thevariant of the invention includes peptides comprising altered amino acidsequences in comparison with a precursor enzyme amino acid sequence(e.g., a wild type or native state enzyme), which peptides retain acharacteristic enzyme nature of the precursor enzyme but which havealtered properties in some specific aspect. For example, an EGIIIvariant may have an increased pH optimum or increased temperature oroxidative stability but will retain cellulolytic activity. It iscontemplated that variants according to the present invention may bederived from a DNA fragment encoding a cellulase derivative wherein thefunctional activity of the expressed cellulase derivative is retained.For example, a DNA fragment encoding a cellulase may further include aDNA sequence or portion thereof encoding a hinge or linker attached tothe cellulase DNA sequence at either the 5′ or 3′ end wherein thefunctional activity of the encoded cellulase domain is retained.

Alignment of Amino Acid Sequences

The variant EGIIIs of this invention have amino acid sequences that arederived from the amino acid sequence of a precursor EGIII. The aminoacid sequence of the EGIII variant differs from the precursor EGIIIamino acid sequence by the substitution, deletion or insertion of one ormore amino acids of the precursor amino acid sequence. In a preferredembodiment, the precursor EGIII is Trichoderma reesei EGIII. The matureamino acid sequence of T. reesei EGIII is shown in FIG. 1. Thus, thisinvention is directed to EGIII variants that contain amino acid residuesat positions that are equivalent to the particular identified residue inT. reesei EGIII as well as at least one residue that is equivalent to anidentified residue in a H. schweinitzii EGIII homolog. A residue (aminoacid) of an EGIII homolog is equivalent to a residue of Trichodermareesei EGIII if it is either homologous (i.e., corresponding in positionin either primary or tertiary structure) or is functionally analogous toa specific residue or portion of that residue in Trichoderma reeseiEGIII (i.e., having the same or similar functional capacity to combine,react, or interact chemically or structurally). As used herein,numbering is intended to correspond to that of the mature EGIII aminoacid sequence as illustrated in FIG. 2. In addition to locations withinthe precursor EGIII, specific residues in the precursor EGIIIcorresponding to the amino acid positions that are responsible forinstability when the precursor EGIII is under thermal or surfactantstress are identified herein for substitution or deletion. The aminoacid position number (e.g., +35) refers to the number assigned to themature Trichoderma reesei EGIII sequence presented in FIG. 1.

The precursor EGIIIs of this invention include naturally occurringcellulases and recombinant cellulases (as defined herein). It isintended that the DNA that encodes the precursor EGIII is modifiedrather than manipulation of the precursor cellulase enzyme per se.Suitable methods for such manipulation of the precursor DNA sequenceinclude methods disclosed herein and in commonly owned U.S. Pat. Nos.4,760,025 and 5,185,258.

Alignment of amino acid sequences to determine homology is preferablydetermined by using a “sequence comparison algorithm.” Optimal alignmentof sequences for comparison can be conducted, e.g., by the localhomology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981),by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443 (1970), by the search for similarity method of Pearson & Lipman,Proc. Nat'l Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection.

An example of an algorithm that is suitable for determining sequencesimilarity is the BLAST algorithm, which is described in Altschul, etal., J. Mol. Biol. 215:403-410 (1990). Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence that eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. These initialneighborhood word hits act as starting points to find longer HSPscontaining them. The word hits are expanded in both directions alongeach of the two sequences being compared for as far as the cumulativealignment score can be increased. Extension of the word hits is stoppedwhen: the cumulative alignment score falls off by the quantity X from amaximum achieved value; the cumulative score goes to zero or below; orthe end of either sequence is reached. The BLAST algorithm parameters W,T, and X determine the sensitivity and speed of the alignment. The BLASTprogram uses as defaults a wordlength (W) of 11, the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915(1989)) alignments (B) of 50, expectation (E) of 10, M′5, N′−4, and acomparison of both strands.

The BLAST algorithm then performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, an amino acid sequence is considered similar to a protease ifthe smallest sum probability in a comparison of the test amino acidsequence to a protease amino acid sequence is less than about 0.1, morepreferably less than about 0.01, and most preferably less than about0.001.

In addition to substitution of an amino acid residue present in H.schweinitzii, other residues can be substituted into EGIII at thermaland/or surfactant sensitive residues. For example, in H.schweinitzii-like EGIII, a serine is at position 35 When this residue issubstituted for the alanine present in T. reesei EGIII, thermalstability decreases by about 4° C. However, if a valine is substitutedfor the alanine, the Tm increases by about 6.5° C. Thus, position 35 isa thermally sensitive site and stability can be increased or decreaseddepending on the substitution.

In addition to modulating thermal and/or surfactant stability, aminoacid substitutions can affect other characteristics of T. reesei EGIII,e.g., substrate binding, inhibitor binding, solubility and performanceunder pH stress. For example, substitution of a tyrosine for atryptophan at position 7 decreases the Tm of an EGIII variant by about1° C. However, this substitution inhibits the binding of cellobiose tothe variant. Cellobiose is an inhibitor of T. reesei EGIII. Thus, thevariant, even though it may be less thermally stable than T. reeseiEGIII, may perform better in applications where cellobiose is present,e.g., biomass conversion.

Additional specific strategies for modifying stability of EGIIIcellulases are provided below:

(1) Increasing the entropy of main-chain unfolding may introducestability to the enzyme. For example, the introduction of prolineresidues into position 2 of reverse turns at the N-termini of α-helicesand in loop structures may significantly stabilize the protein byincreasing the entropy of the unfolding (see, e.g., Watanabe, et al.,Eur. J. Biochem. 226:277-283 (1994)). Similarly, glycine residues haveno β-carbon, and thus have considerably greater backbone conformationalfreedom than many other residues. This may lead to high flexibility withresultant weak stability. Replacement of glycines preferably withalanines, may reduce the flexibility and improve stability.Additionally, by shortening external loops it may be possible to improvestability. It has been observed that hyperthermophile produced proteinshave shorter external loops than their mesophilic homologues (see, e.g.,Russel, et al., Current Opinions in Biotechnology 6:370-374 (1995)). Theintroduction of disulfide bonds may also be effective to stabilizedistinct tertiary structures in relation to each other. Thus, theintroduction of cysteines at residues accessible to existing cysteinesor the introduction of pairs of cysteines that could form disulfidebonds would alter the stability of an EGIII variant.

(2) Decreasing internal cavities by increasing side-chain hydrophobicitymay alter the stability of an enzyme. Reducing the number and volume ofinternal cavities increases the stability of enzyme by maximizinghydrophobic interactions and reducing packing defects (see, e.g.,Matthews, Ann. Rev. Biochem. 62:139-160 (1993); Burley, et al., Science229:23-29 (1985); Zuber, Biophys. Chem. 29:171-179 (1988); Kellis, etal., Nature 333:784-786 (1988)). It is known that multimeric proteinsfrom thermophiles often have more hydrophobic sub-unit interfaces withgreater surface complementarity than their mesophilic counterparts(Russel, et al., supra). This principle is believed to be applicable todomain interfaces of monomeric proteins. Specific substitutions that mayimprove stability by increasing hydrophobicity include lysine toarginine, serine to alanine and threonine to alanine (Russel, et al.,supra). Modification by substitution to alanine or proline may increaseside-chain size with resultant reduction in cavities, better packing andincreased hydrophobicity. Substitutions to reduce the size of thecavity, increase hydrophobicity and improve the complementarity theinterfaces between the domains of EGIII may improve stability of theenzyme. Specifically, modification of the specific residue at thesepositions with a different residue selected from any of phenylalanine,tryptophan, tyrosine, leucine and isoleucine may improve performance.

(3) Balancing charge in rigid secondary structure, i.e., α-helices andβ-turns may improve stability. For example, neutralizing partialpositive charges on a helix N-terminus with negative charge on asparticacid may improve stability of the structure (see, e.g., Eriksson, etal., Science 255:178-183 (1992)). Similarly, neutralizing partialnegative charges on helix C-terminus with positive charge may improvestability. Removing positive charge from interacting with peptideN-terminus in β-turns should be effective in conferring tertiarystructure stability. Substitution with a non-positively charged residuecould remove an unfavorable positive charge from interacting with anamide nitrogen present in a turn.

(4) Introducing salt bridges and hydrogen bonds to stabilize tertiarystructures may be effective. For example, ion pair interactions, e.g.,between aspartic acid or glutamic acid and lysine, arginine orhistidine, may introduce strong stabilizing effects and may be used toattach different tertiary structure elements with a resultantimprovement in thermostability. Additionally, increases in the number ofcharged residue/non-charged residue hydrogen bonds, and the number ofhydrogen-bonds generally, may improve thermostability (see, e.g.,Tanner, et al., Biochemistry 35:2597-2609). Substitution with asparticacid, asparagine, glutamic acid or glutamine may introduce a hydrogenbond with a backbone amide. Substitution with arginine may improve asalt bridge and introduce an H-bond into a backbone carbonyl.

(5) Avoiding thermolabile residues in general may increase thermalstability. For example, asparagine and glutamine are susceptible todeamidation and cysteine is susceptible to oxidation at hightemperatures. Reducing the number of these residues in sensitivepositions may result in improved thermostability (Russel, et al.,supra). Substitution or deletion by any residue other than glutamine orcysteine may increase stability by avoidance of a thermo stabileresidue.

(6) Stabilization or destabilization of binding of a ligand that confersmodified stability to EGIII variants. For example, a component of thematrix in which the EGIII variants of this invention are used may bindto a specific surfactant/thermal sensitivity site of the EGIII variant.By modifying the site through substitution, binding of the component tothe variant may be strengthened or diminished. For example, anon-aromatic residue in the binding crevice of EGIII may be substitutedwith phenylalanine or tyrosine to introduce aromatic side-chainstabilization where interaction of the cellulose substrate may interactfavorably with the benzyl rings, increasing the stability of the EGIIIvariant.

(8) Increasing the electronegativity of any of the surfactant/thermalsensitivity ligands may improve stability under surfactant or thermalstress. For example, substitution with phenylalanine or tyrosine mayincrease the electronegativity of D residues by improving shielding fromsolvent, thereby improving stability.

Variant EGIII

The present invention relates to the expression, purification and/orisolation and use of variant EGIII. These enzymes are preferablyprepared by recombinant methods utilizing the gene identified andisolated according to the methods described below. However, enzymes foruse in the present invention may be obtained by other art-recognizedmeans such as purification from natural isolates.

Techniques that can be used to isolate EGIII encoding DNA sequences iswell known in the art and include, but are not limited to, cDNA and/orgenomic library screening with a homologous DNA probe and expressionscreening with activity assays or antibodies against EGIII. Any of thesemethods can be found in Sambrook, et al. or in Current Protocols inMolecular Biology, F. Ausubel, et al., ed. Greene Publishing andWiley-Interscience, New York (1987) (“Ausubel”).

After the isolation and cloning of the EGIII, other methods known in theart, such as site directed mutagenesis, are used to make thesubstitutions, additions or deletions that correspond to substitutedamino acids in the expressed EGIII variant. Again, site directedmutagenesis and other methods of incorporating amino acid changes inexpressed proteins at the DNA level can be found in Sambrook, et al. andAusubel, et al.

After DNA sequences that encode the EGIII variants have been cloned intoDNA constructs, the DNA is used to transform microorganisms. Themicroorganism to be transformed for the purpose of expressing a variantEGIII according to the present invention may advantageously comprise astrain derived from Trichoderma sp. Thus, a preferred mode for preparingvariant EGIII cellulases according to the present invention comprisestransforming a Trichoderma sp. host cell with a DNA construct comprisingat least a fragment of DNA encoding a portion or all of the variantEGIII. The DNA construct will generally be functionally attached to apromoter. The transformed host cell is then grown under conditions so asto express the desired protein. Subsequently, the desired proteinproduct is purified to substantial homogeneity.

However, it may in fact be that the best expression vehicle for a givenDNA encoding a variant EGIII may differ from T. reesei. Thus, it may bethat it will be most advantageous to express a protein in atransformation host which bears phylogenetic similarity to the sourceorganism for the variant EGIII. Accordingly, the present description ofa Trichoderma spp. expression system is provided for illustrativepurposes only and as one option for expressing the variant EGIII of theinvention. One of skill in the art, however, may be inclined to expressthe DNA encoding variant EGIII in a different host cell if appropriateand it should be understood that the source of the variant EGIII shouldbe considered in determining the optimal expression host. For example,Aspergillus niger can be used as an expression host. See, WO 98/31821for a description of transformation into A. niger Additionally, theskilled worker in the field will be capable of selecting the bestexpression system for a particular gene through routine techniquesutilizing the tools available in the art.

In one embodiment, the strain comprises T. reesei (reesei) which is auseful strain for obtaining overexpressed protein. For example, RL-P37,described by Sheir-Neiss, et al., Appl. Microbiol. Biotechnol. 20:46-53(1984) is known to secrete elevated amounts of cellulase enzymes.Functional equivalents of RL-P37 include Trichoderma reesei (reesei)strain RUT-C30 (ATCC No. 56765) and strain QM9414 (ATCC No. 26921). Itis contemplated that these strains would also be useful inoverexpressing variant EGIII.

Where it is desired to obtain the variant EGIII in the absence ofpotentially detrimental native cellulolytic activity, it is useful toobtain a Trichoderma host cell strain which has had one or morecellulase genes deleted prior to introduction of a DNA construct orplasmid containing the DNA fragment encoding the variant EGIII. Suchstrains may be prepared by the method disclosed in U.S. Pat. No.5,246,853 and WO 92/06209, which disclosures are hereby incorporated byreference. By expressing a variant EGIII cellulase in a hostmicroorganism that is missing one or more cellulase genes, theidentification and subsequent purification procedures are simplified.Any gene from Trichodermia sp. which has been cloned can be deleted, forexample, the cbh1, cbh2, egl1, and egl3 genes as well as those encodingEGIII and/or EGV protein (see e.g., U.S. Pat. No. 5,475,101 and WO94/28117, respectively).

Gene deletion may be accomplished by inserting a form of the desiredgene to be deleted or disrupted into a plasmid by methods known in theart. The deletion plasmid is then cut at an appropriate restrictionenzyme site(s), internal to the desired gene coding region, and the genecoding sequence or part thereof replaced with a selectable marker.Flanking DNA sequences from the locus of the gene to be deleted ordisrupted, preferably between about 0.5 to 2.0 kb, remain on either sideof the selectable marker gene. An appropriate deletion plasmid willgenerally have unique restriction enzyme sites present therein to enablethe fragment containing the deleted gene, including flanking DNAsequences, and the selectable marker gene to be removed as a singlelinear piece.

A selectable marker must be chosen so as to enable detection of thetransformed microorganism. Any selectable marker gene that is expressedin the selected microorganism will be suitable. For example, withTrichoderma sp., the selectable marker is chosen so that the presence ofthe selectable marker in the transformants will not significantly affectthe properties thereof. Such a selectable marker may be a gene whichencodes an assayable product. For example, a functional copy of aTrichoderma sp. gene may be used which if lacking in the host strainresults in the host strain displaying an auxotrophic phenotype.

In a preferred embodiment, a pyr4⁻ derivative strain of Trichoderma sp.is transformed with a functional pyr4 gene, which thus provides aselectable marker for transformation. A pyr4⁻ derivative strain may beobtained by selection of Trichoderma sp. strains that are resistant tofluoroorotic acid (FOA). The pyr4 gene encodesorotidine-5′-monophosphate decarboxylase, an enzyme required for thebiosynthesis of uridine. Strains with an intact pyr4 gene grow in amedium lacking uridine but are sensitive to fluoroorotic acid. It ispossible to select pyr4⁻ derivative strains that lack a functionalorotidine monophosphate decarboxylase enzyme and require uridine forgrowth by selecting for FOA resistance. Using the FOA selectiontechnique it is also possible to obtain uridine-requiring strains whichlack a functional orotate pyrophosphoribosyl transferase. It is possibleto transform these cells with a functional copy of the gene encodingthis enzyme (Berges & Barreau, Curr. Genet. 19:359-365 (1991)).Selection of derivative strains is easily performed using the FOAresistance technique referred to above, and thus, the pyr4 gene ispreferably employed as a selectable marker.

To transform pyr4⁻ Trichoderma sp. so as to be lacking in the ability toexpress one or more cellulase genes, a single DNA fragment comprising adisrupted or deleted cellulase gene is then isolated from the deletionplasmid and used to transform an appropriate pyr⁻ Trichoderma host.Transformants are then identified and selected based on their ability toexpress the pyr4 gene product and thus compliment the uridine auxotrophyof the host strain. Southern blot analysis is then carried out on theresultant transformants to identify and confirm a double crossoverintegration event that replaces part or all of the coding region of thegenomic copy of the gene to be deleted with the pyr4 selectable markers.

Although the specific plasmid vectors described above relate topreparation of pyr⁻ transformants, the present invention is not limitedto these vectors. Various genes can be deleted and replaced in theTrichoderma sp. strain using the above techniques. In addition, anyavailable selectable markers can be used, as discussed above. In fact,any Trichoderma sp. gene that has been cloned, and thus identified, canbe deleted from the genome using the above-described strategy.

As stated above, the host strains used are derivatives of Trichodermasp. that lack or have a nonfunctional gene or genes corresponding to theselectable marker chosen. For example, if the selectable marker of pyr4is chosen, then a specific pyr4⁻ derivative strain is used as arecipient in the transformation procedure. Similarly, selectable markerscomprising Trichoderma sp. genes equivalent to the Aspergillus nidulansgenes amdS, argB, trpC, niaD may be used. The corresponding recipientstrain must therefore be a derivative strain such as argB⁻, trpC⁻,niaD⁻, respectively.

DNA encoding the variant EGIII cellulase is then prepared for insertioninto an appropriate microorganism. According to the present invention,DNA encoding a variant EGIII cellulase comprises the DNA necessary toencode for a protein that has functional cellulolytic activity. The DNAfragment or DNA variant fragment encoding the variant EGIII cellulase orderivative may be functionally attached to a fungal promoter sequence,for example, the promoter of the cbh1 or egl1 gene.

It is also contemplated that more than one copy of DNA encoding avariant EGIII cellulase may be recombined into the strain to facilitateoverexpression. The DNA encoding the variant EGIII cellulase may beprepared by the construction of an expression vector carrying the DNAencoding the cellulase. The expression vector carrying the inserted DNAfragment encoding the variant EGIII cellulase may be any vector which iscapable of replicating autonomously in a given host organism or ofintegrating into the DNA of the host, typically a plasmid. In preferredembodiments two types of expression vectors for obtaining expression ofgenes are contemplated. The first contains DNA sequences in which thepromoter, gene-coding region, and terminator sequence all originate fromthe gene to be expressed. Gene truncation may be obtained where desiredby deleting undesired DNA sequences (e.g., coding for unwanted domains)to leave the domain to be expressed under control of its owntranscriptional and translational regulatory sequences. A selectablemarker is also contained on the vector allowing the selection forintegration into the host of multiple copies of the novel genesequences.

The second type of expression vector is preassembled and containssequences required for high-level transcription and a selectable marker.It is contemplated that the coding region for a gene or part thereof canbe inserted into this general-purpose expression vector such that it isunder the transcriptional control of the expression cassettes promoterand terminator sequences. For example, pTEX is such a general-purposeexpression vector. Genes or part thereof can be inserted downstream ofthe strong cbh1 promoter.

In the vector, the DNA sequence encoding the variant EGIII cellulase ofthe present invention should be operably linked to transcriptional andtranslational sequences, i.e., a suitable promoter sequence and signalsequence in reading frame to the structural gene. The promoter may beany DNA sequence that shows transcriptional activity in the host celland may be derived from genes encoding proteins either homologous orheterologous to the host cell. The signal peptide provides forextracellular production of the variant EGIII cellulase or derivativesthereof. The DNA encoding the signal sequence is preferably that whichis naturally associated with the gene to be expressed, however thesignal sequence from any suitable source, for example anexo-cellobiohydrolase or endoglucanase from Trichoderma, is contemplatedin the present invention.

The procedures used to ligate the DNA sequences coding for the ariantEGIII cellulase of the present invention with the promoter, andinsertion into suitable vectors are well known in the art.

The DNA vector or construct described above may be introduced in thehost cell in accordance with known techniques such as transformation,transfection, microinjection, microporation, biolistic bombardment andthe like.

In the preferred transformation technique, it must be taken into accountthat the permeability of the cell wall to DNA in Trichodemina sp. isvery low. Accordingly, uptake of the desired DNA sequence, gene or genefragment is at best minimal. There are a number of methods to increasethe permeability of the Trichoderma sp. cell wall in the derivativestrain (i.e., lacking a functional gene corresponding to the usedselectable marker) prior to the transformation process.

The preferred method in the present invention to prepare Trichoderma sp.for transformation involves the preparation of protoplasts from fungalmycelium. The mycelium can be obtained from germinated vegetativespores. The mycelium is treated with an enzyme that digests the cellwall resulting in protoplasts. The protoplasts are then protected by thepresence of an osmotic stabilizer in the suspending medium. Thesestabilizers include sorbitol, mannitol, potassium chloride, magnesiumsulfate and the like. Usually the concentration of these stabilizersvaries between 0.8 M and 1.2 M. It is preferable to use about a 1.2 Msolution of sorbitol in the suspension medium.

Uptake of the DNA into the host Trichodemina sp. strain is dependentupon the calcium ion concentration. Generally between about 10 mM CaCl₂and 50 mM CaCl₂ is used in an uptake solution. Besides the need for thecalcium ion in the uptake solution, other items generally included are abuffering system such as TE buffer (10 Mm Tris, pH 7.4; 1 mM EDTA) or 10mM MOPS, pH 6.0 buffer (morpholinepropanesulfonic acid) and polyethyleneglycol (PEG). It is believed that the polyethylene glycol acts to fusethe cell membranes thus permitting the contents of the medium to bedelivered into the cytoplasm of the Trichoderma sp. strain and theplasmid DNA is transferred to the nucleus. This fusion frequently leavesmultiple copies of the plasmid DNA tenderly integrated into the hostchromosome.

Usually a suspension containing the Trichoderma sp. protoplasts or cellsthat have been subjected to a permeability treatment at a density of 10⁸to 10⁹/mL, preferably 2×10⁸/mL are used in transformation. A volume of100 μL of these protoplasts or cells in an appropriate solution (e.g.,1.2 M sorbitol; 50 mM CaCl₂) are mixed with the desired DNA. Generally ahigh concentration of PEG is added to the uptake solution. From 0.1 to 1volume of 25% PEG 4000 can be added to the protoplast suspension.However, it is preferable to add about 0.25 volumes to the protoplastsuspension. Additives such as dimethyl sulfoxide, heparin, spermidine,potassium chloride and the like may also be added to the uptake solutionand aid in transformation.

Generally, the mixture is then incubated at approximately 0° C. for aperiod of between 10 to 30 minutes. Additional PEG is then added to themixture to further enhance the uptake of the desired gene or DNAsequence. The 25% PEG 4000 is generally added in volumes of 5 to 15times the volume of the transformation mixture; however, greater andlesser volumes may be suitable. The 25% PEG 4000 is preferably about 10times the volume of the transformation mixture. After the PEG is added,the transformation mixture is then incubated at room temperature beforethe addition of a sorbitol and CaCl₂ solution. The protoplast suspensionis then further added to molten aliquots of a growth medium. This growthmedium permits the growth of transformants only. Any growth medium canbe used in the present invention that is suitable to grow the desiredtransformants. However, if Pyr⁺ transformants are being selected it ispreferable to use a growth medium that contains no uridine. Thesubsequent colonies are transferred and purified on a growth mediumdepleted of uridine.

At this stage, stable transformants may be distinguished from unstabletransformants by their faster growth rate and the formation of circularcolonies with a smooth, rather than ragged outline on solid culturemedium lacking uridine. Additionally, in some cases a further test ofstability may made by growing the transformants on solid non-selectivemedium (i.e. containing uridine), harvesting spores from this culturemedium and determining the percentage of these spores which willsubsequently germinate and grow on selective medium lacking uridine.

In a particular.embodiment of the above method, the variant EGIIIcellulases or derivatives thereof are recovered in active form from thehost cell after growth in liquid media either as a result of theappropriate post translational processing of the novel variant EGIIIcellulase or derivatives thereof.

The expressed variant EGIII cellulase may be recovered from the mediumby conventional techniques including separations of the cells from themedium by centrifugation, filtration, and precipitation of the proteinsin the supernatant or filtrate with a salt, for example, ammoniumsulfate. Additionally, chromatography procedures such as ion exchangechromatography or affinity chromatography may be used. Antibodies(polyclonal or monoclonal) may be raised against the natural purifiedvariant EGIII cellulase, or synthetic peptides may be prepared fromportions of the variant EGIII cellulase molecule and used to raisepolyclonal antibodies.

Compositions Comprising the EGIII Variants of This Invention

Treatment of textiles according to the present invention contemplatestextile processing or cleaning with a composition comprising acellulase. Such treating includes, but is not limited to, stonewashing,modifying the texture, feel and/or appearance of cellulose containingfabrics or other techniques used during manufacturing orcleaning/reconditioning of cellulose containing fabrics. Additionally,treating within the context of this invention contemplates the removalof “immature” or “dead” cotton, from cellulosic fabric or fibers.Immature cotton is significantly more amorphous than mature cotton andresults in a lesser quality fabric when present due to, for example,uneven dyeing. The composition contemplated in the present inventionfurther includes a cellulase component for use in washing of a soiledmanufactured cellulose containing fabric. For example, the cellulase maybe used in a detergent composition for washing laundry. Detergentcompositions useful in accordance with the present invention includespecial formulations such as pre-wash, pre-soak and home-use colorrestoration compositions. Such treating compositions, as describedherein, may be in the form of a concentrate which requires dilution orin the form of a dilute solution or form which can be applied directlyto the cellulose containing fabric. General treatment techniques forcellulase treatment of textiles are described in, for example, EPPublication No. 220 016 and GB Application Nos. 1,368,599 and 2,095,275.

Treatment of a cellulosic material according to the present inventionfurther contemplates the treatment of animal feed, pulp and/or paper,food and grain for purposes known in the art. For example, cellulase isknown to increase the value of animal feed, improve the drainability ofwood pulp, enhance food products and reduce fiber in grain during thegrain wet milling process or dry milling process.

Treating according to the instant invention comprises preparing anaqueous solution that contains an effective amount of cellulase togetherwith other optional ingredients including, for example, a buffer, asurfactant, and/or a scouring agent. An effective amount of cellulaseenzyme composition is a concentration of cellulase enzyme sufficient forits intended purpose. Thus, for example, an “effective amount” ofcellulase in a stonewashing composition according to the presentinvention is that amount which will provide the desired effect, e.g., toproduce a worn and faded look in the seams and on fabric panels.Similarly, an “effective amount” of cellulase in a composition intendedfor improving the feel and/or appearance of a cellulose containingfabric is that amount which will produce measurable improvements in thefeel, e.g., improving the smoothness of the fabric, or appearance, e.g.,removing pills and fibrils which tend to reduce the sharpness inappearance of a fabric. The amount of cellulase employed is alsodependent on the equipment employed, the process parameters employed(the temperature of the cellulase treatment solution, the exposure timeto the cellulase solution, and the like), and the cellulase activity(e.g., a particular solution will require a lower concentration ofcellulase where a more active cellulase composition is used as comparedto a less active cellulase composition). The exact concentration ofcellulase in the aqueous treatment solution to which the fabric to betreated is added can be readily determined by the skilled artisan basedon the above factors as well as the desired result. In stonewashingprocesses, it has generally been preferred that the cellulase be presentin the aqueous treating solution in a concentration of from about 0.5 to5,000 ppm and most preferably about 10 to 200 ppm total protein. Incompositions for the improvement of feel and/or appearance of acellulose containing fabric, it has generally been preferred that thecellulase be present in the aqueous treating solution in a concentrationof from about 0.1 to 2000 ppm and most preferably about 0.5 to 200 ppmtotal protein.

In a preferred treating embodiment, a buffer is employed in the treatingcomposition such that the concentration of buffer is sufficient tomaintain the pH of the solution within the range wherein the employedcellulase exhibits activity which, in turn, depends on the nature of thecellulase employed. The exact concentration of buffer employed willdepend on several factors that the skilled artisan can readily take intoaccount. For example, in a preferred embodiment, the buffer, as well asthe buffer concentration, is selected so as to maintain the pH of thefinal cellulase solution within the pH range required for optimalcellulase activity. The determination of the optimal pH range of thecellulases of the invention can be ascertained according to well-knowntechniques. Suitable buffers at pH within the activity range of thecellulase are well known to those skilled in the art in the field.

In addition to cellulase and a buffer, the treating composition mayoptionally contain a surfactant. Suitable surfactants include anysurfactant compatible with the cellulase and the fabric including, forexample, anionic, non-ionic and ampholytic surfactants. Suitable anionicsurfactants for use herein include linear or branchedalkylbenzenesulfonates; alkyl or alkenyl ether sulfates having linear orbranched alkyl groups or alkenyl groups; alkyl or alkenyl sulfates;olefinsulfonates; alkanesulfonates and the like. Suitable counter ionsfor anionic surfactants include alkali metal ions such as sodium andpotassium; alkaline earth metal ions such as calcium and magnesium;ammonium ion; and alkanolamines having 1 to 3 alkanol groups of carbonnumber 2 or 3. Ampholytic surfactants include quaternary ammonium saltsulfonates, and betaine-type ampholytic surfactants. Such ampholyticsurfactants have both the positive and negative charged groups in thesame molecule. Nonionic surfactants generally comprise polyoxyalkyleneethers, as well as higher fatty acid alkanolamides or alkylene oxideadduct thereof, and fatty acid glycerine monoesters. Mixtures ofsurfactants can also be employed in manners known to those killed in theart.

A concentrated cellulase composition can be prepared for use in themethods described herein. Such concentrates contain concentrated amountsof the cellulase composition described above, buffer and surfactant,preferably in an aqueous solution. When so formulated, the cellulaseconcentrate can readily be diluted with water so as to quickly andaccurately prepare cellulase preparations having the requisiteconcentration of each constituent. When aqueous concentrates areformulated, these concentrates can be diluted so as to arrive at therequisite concentration of the components in the cellulase solution asindicated above. As is readily apparent, such cellulase concentrateswill permit facile formulation of the cellulase solutions as well aspermit feasible transportation of the composition to the location whereit will be used. The treating concentrate can be in any art recognizedform, for example, liquid, emulsion, gel, or paste. Such forms are wellknown to those skilled in the art.

When a solid cellulase concentrate is employed, the cellulasecomposition may be a granule, a powder, an agglomerate or a solid disk.The granules can be formulated so as to contain materials to reduce therate of dissolution of the granules into the wash medium. Such materialsand granules are disclosed in U.S. Pat. No. 5,254,283, which isincorporated herein by reference in its entirety.

Other materials can also be used with or placed in the cellulasecomposition of the present invention as desired, including stones,pumice, fillers, solvents, enzyme activators, and anti-redepositionagents depending on the eventual use of the composition.

By way of example, stonewashing methods will be described in detail,however, the parameters described are readily modified by the skilledartisan for other applications, e.g., improving the feel and/orappearance of a fabric. The cellulose containing fabric is contactedwith the cellulase containing stonewashing composition containing aneffective amount of the cellulase by intermingling the treatingcomposition with the stonewashing composition, and thus bringing thecellulase enzyme into proximity with the fabric. Subsequently, theaqueous solution containing the cellulase and the fabric is agitated. Ifthe treating composition is an aqueous solution, the fabric may bedirectly soaked in the solution. Similarly, where the stonewashingcomposition is a concentrate, the concentrate is diluted into a waterbath with the cellulose containing fabric. When the stonewashingcomposition is in a solid form, for example a pre-wash gel or solidstick, the stonewashing composition may be contacted by directlyapplying the composition to the fabric or to the wash liquor.

The cellulose containing fabric is incubated with the stonewashingsolution under conditions effective to allow the enzymatic action toconfer a stonewashed appearance to the cellulose containing fabric. Forexample, during stonewashing, the pH, liquor ratio, temperature andreaction time may be adjusted to optimize the conditions under which thestonewashing composition acts. “Effective conditions” necessarily refersto the pH, liquor ratio, and temperature that allow the cellulase enzymeto react efficiently with cellulose containing fabric, in this case toproduce the stonewashed effect. However, such conditions are readilyascertainable by one of skill in the art. The reaction conditionseffective for the stonewashing compositions of the present invention aresubstantially similar to well known methods used with correspondingprior art cellulase compositions. Accordingly, it is within the skill ofthose in the art to maximize conditions for using the stonewashingcompositions according to the present invention.

The liquor ratios during stonewashing, i.e., the ratio of weight ofstonewashing composition solution (i.e., the wash liquor) to the weightof fabric, employed herein is generally an amount sufficient to achievethe desired stonewashing effect in the denim fabric and is dependentupon the process used. Preferably, the liquor ratios are from about 4:1to about 50:1; more preferably from about 5:1 to about 20:1, and mostpreferably from about 10:1 to about 15:1.

Reaction temperatures during stonewashing with the present stonewashingcompositions are governed by two competing factors. Firstly, highertemperatures generally correspond to enhanced reaction kinetics, i.e.,faster reactions, which permit reduced reaction times as compared toreaction times required at lower temperatures. Accordingly, reactiontemperatures are generally at least about 10° C. and greater. Secondly,cellulase is a protein which loses activity beyond a given reactiontemperature, which temperature is dependent on the nature of thecellulase used. Thus, if the reaction temperature is permitted to go toohigh, the cellulolytic activity is lost as a result of the denaturing ofthe cellulase. While standard temperatures for cellulase usage in theart are generally in the range of 35° C. to 65° C., which conditionswould also be expected to be suitable for the cellulase of theinvention, the optimal temperature conditions should be ascertainedaccording to well known techniques with respect to the specificcellulase used.

Reaction times are dependent on the specific conditions under which thestonewashing occurs. For example, pH, temperature and concentration ofcellulase will all affect the optimal reaction time. Generally, reactiontimes are from about 5 minutes to about 5 hours, and preferably fromabout 10 minutes to about 3 hours and, more preferably, from about 20minutes to about 1 hour. According to yet another preferred embodimentof the present invention, the cellulase of the invention may be employedin a detergent composition. The detergent compositions according to thepresent invention are useful as pre-wash compositions, pre-soakcompositions, or for cleaning during the regular wash or rinse cycle.Preferably, the detergent composition of the present invention comprisesan effective amount of cellulase, a surfactant, and optionally includesother ingredients described below.

An effective amount of cellulase employed in the detergent compositionsof this invention is an amount sufficient to impart the desirableeffects known to be produced by cellulase on cellulose containingfabrics, for example, depilling, softening, anti-pilling, surface fiberremoval, anti-graying and cleaning. Preferably, the cellulase in thedetergent composition is employed in a concentration of from about 10ppm to about 20,000 ppm of detergent.

The concentration of cellulase enzyme employed in the detergentcomposition is preferably selected so that upon dilution into a washmedium, the concentration of cellulase enzyme is in a range of about0.01 to about 1000 ppm, preferably from about 0.02 ppm to about 500 ppm,and most preferably from about 0.5 ppm to about 250 ppm total protein.The amount of cellulase enzyme employed in the detergent compositionwill depend on the extent to which the detergent will be diluted uponaddition to water so as to form a wash solution.

The detergent compositions of the present invention may be in any artrecognized form, for example, as a liquid, in granules, in emulsions, ingels, or in pastes. Such forms are well known to the skilled artisan.When a solid detergent composition is employed, the cellulase ispreferably formulated as granules. Preferably, the granules can beformulated so as to additionally contain a cellulase-protecting agent.The granule can be formulated so as to contain materials to reduce therate of dissolution of the granule into the wash medium. Such materialsand granules are disclosed in U.S. Pat. No. 5,254,283, which isincorporated herein by reference in its entirety.

The detergent compositions of this invention employ a surface-activeagent, e.g., surfactant, including anionic, non-ionic and ampholyticsurfactants well known for their use in detergent compositions. Inaddition to the cellulase composition and the surfactant(s), thedetergent compositions of this invention can optionally contain one ormore of the following components.

Hydrolases Except Cellulase

Suitable hydrolases include carboxylate ester hydrolase, thioesterhydrolase, phosphate monoester hydrolase, and phosphate diesterhydrolase which act on the ester bond; glycoside hydrolase which acts onglycosyl compounds; an enzyme that hydrolyzes N-glycosyl compounds;thioether hydrolase which acts on the ether bond; anda-amino-acyl-peptide hydrolase, peptidyl-amino acid hydrolase,acyl-amino acid hydrolase, dipeptide hydrolase, and peptidyl-peptidehydrolase which act on the peptide bond. Preferable among them arecarboxylate ester hydrolase, glycoside hydrolase, and peptidyl-peptidehydrolase. Suitable hydrolases include (1) proteases belonging topeptidyl-peptide hydrolase such as pepsin, pepsin B, rennin, trypsin,chymotrypsin A, chymotrypsin B, elastase, enterokinase, cathepsin C,papain, chymopapain, ficin, thrombin, fibrinolysin, renin, subtilisin,aspergillopeptidase A, collagenase, clostridiopeptidase B, kallikrein,gastrisin, cathepsin D., bromelin, keratinase, chymotrypsin C, pepsin C,aspergillopeptidase B, urokinase, carboxypeptidase A and B, andaminopeptidase; (2) glycoside hydrolases (cellulase which is anessential ingredient is excluded from this group) α-amylase, β-amylase,gluco amylase, invertase, lysozyme, pectinase, chitinase, anddextranase. Preferably among them are α-amylase and β-amylase. Theyfunction in acid to neutral systems, but one which is obtained frombacteria exhibits high activity in an alkaline system; (3) carboxylateester hydrolase including carboxyl esterase, lipase, pectin esterase,and chlorophyllase. Especially effective among them is lipase.

The hydrolase other than cellulase is incorporated into the detergentcomposition as much as required according to the purpose. It shouldpreferably be incorporated in an amount of 0.001 to 5 weight percent,and more preferably 0.02 to 3 weight percent, in terms of purifiedprotein. This enzyme should be used in the form of granules made ofcrude enzyme alone or in combination with other components in thedetergent composition. Granules of crude enzyme are used in such anamount that the purified enzyme is 0.001 to 50 weight percent in thegranules. The granules are used in an amount of 0.002 to 20 andpreferably 0.1 to 10 weight percent. As with cellulases, these granulescan be formulated so as to contain an enzyme protecting agent and adissolution retardant material.

Builders

A. Divalent Sequestering Agents.

The composition may contain from about 0 to about 50 weight percent ofone or more builder components selected from the group consisting ofalkali metal salts and alkanolamine salts of the following compounds:phosphates, phosphonates, phosphonocarboxylates, salts of amino acids,aminopolyacetates high molecular electrolytes, non-dissociatingpolymers, salts of dicarboxylic acids, and aluminosilicate salts.Suitable divalent sequestering gents are disclosed in British PatentApplication No. 2 094 826 A, the disclosure of which is incorporatedherein by reference.

B. Alkalis or Inorganic Electrolytes

The composition may contain from about 1 to about 50 weight percent,preferably from about 5 to about 30 weight percent, based on thecomposition of one or more alkali metal salts of the following compoundsas the alkalis or inorganic electrolytes: silicates, carbonates andsulfates as well as organic alkalis such as triethanolamine,diethanolamine, monoethanolamine and triisopropanolamine.

Antiredeposition Agents

The composition may contain from about 0.1 to about 5 weight percent ofone or more of the following compounds as antiredeposition agents:polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone andcarboxymethylcellulose.

Among them, a combination of carboxymethyl-cellulose and/or polyethyleneglycol with the cellulase composition of the present invention providesfor an especially useful dirt removing composition.

Bleaching Agents

The use of the cellulase of the present invention in combination with ableaching agent such as potassium monopersulfate, sodium percarbonate,sodium perborate, sodium sulfate/hydrogen peroxide adduct and sodiumchloride/hydrogen peroxide adduct or/and a photo-sensitive bleaching dyesuch as zinc or aluminum salt of sulfonated phthalocyanine furtherimproves the detergenting effects. Similarly, bleaching agents andbleach catalysts as described in EP 684 304 may be used.

Bluing Agents and Fluorescent Dyes

Various bluing agents and fluorescent dyes may be incorporated in thecomposition, if necessary. Suitable bluing agents and fluorescent dyesare disclosed in British Patent Application No. 2 094 826 A, thedisclosure of which is incorporated herein by reference.

Caking Inhibitors

The following caking inhibitors may be incorporated in the powderydetergent: p-toluenesulfonic acid salts, xylenesulfonic acid salts,acetic acid salts, sulfosuccinic acid salts, talc, finely pulverizedsilica, amorphous silicas, clay, calcium silicate (such as Micro-Cell ofJohns Manville Co.), calcium carbonate and magnesium oxide.

Masking Agents for Factors Inhibiting the Cellulase Activity

The cellulase composition of this invention is deactivated in some casesin the presence of copper, zinc, chromium, mercury, lead, manganese orsilver ions or their compounds. Various metal chelating agents andmetal-precipitating agents are effective against these inhibitors. Theyinclude, for example, divalent metal ion sequestering agents as listedin the above item with reference to optional additives as well asmagnesium silicate and magnesium sulfate.

Cellobiose, glucose and gluconolactone act sometimes as inhibitors. Itis preferred to avoid the co-presence of these saccharides with thecellulase as far as possible. In case the co-presence in unavoidable, itis necessary to avoid the direct contact of the saccharides with thecellulase by, for example, coating them.

Long-chain-fatty acid salts and cationic surfactants act as theinhibitors in some cases. However, the co-presence of these substanceswith the cellulase is allowable if the direct contact of them isprevented by some means such as tableting or coating.

The above-mentioned masking agents and methods may be employed, ifnecessary, in the present invention.

Cellulase-Activators

The activators may vary depending on the specific cellulase. In thepresence of proteins, cobalt and its salts, magnesium and its salts, andcalcium and its salts, potassium and its salts, sodium and its salts ormonosaccharides such as mannose and xylose, many cellulases areactivated and their deterging powers are improved remarkably.

Antioxidants

The antioxidants include, for example, tert-butyl-hydroxytoluene,4,4′-butylidenebis(6-tert-butyl-3-methylphenol),2,2′-butylidenebis(6-tert-butyl-4-methylphenol), monostyrenated cresol,distyrenated cresol, monostyrenated phenol, distyrenated phenol and1,1-bis(4-hydroxy-phenyl)cyclohexane.

Solubilizers

The solubilizers include, for example, lower alcohols such as ethanol,benzenesulfonate salts, lower alkylbenzenesulfonate salts such asp-toluenesulfonate salts, glycols such as propylene glycol,acetylbenzene-sulfonate salts, acetamides, pyridinedicarboxylic acidamides, benzoate salts and urea.

The detergent composition of the present invention can be used in abroad pH range from acidic to alkaline pH. In a preferred embodiment,the detergent composition of the present invention can be used in mildlyacidic, neutral or alkaline detergent wash media having a pH of fromabove 5 to no more than about 12.

Aside from the above ingredients, perfumes, buffers, preservatives,dyes, and the like can be used, if desired, with the detergentcompositions of this invention. Such components are conventionallyemployed in amounts heretofore used in the art.

When a detergent base used in the present invention is in the form of apowder, it may be one that is prepared by any known preparation methodsincluding a spray-drying method and a granulation method. The detergentbase obtained particularly by the spray-drying method, agglomerationmethod, dry mixing method or non-tower route methods are preferred. Thedetergent base obtained by the spray-drying method is not restrictedwith respect to preparation conditions. The detergent base obtained bythe spray-drying method is hollow granules which are obtained byspraying an aqueous slurry of heat-resistant ingredients, such assurface active agents and builders, into a hot space. After thespray-drying, perfumes, enzymes, bleaching agents, inorganic alkalinebuilders may be added. With a highly dense, granular detergent baseobtained such as by the spray-drying-granulation or agglomerationmethod, various ingredients may also be added after the preparation ofthe base.

When the detergent base is a liquid, it may be either a homogeneoussolution or an inhomogeneous dispersion. For removing the decompositionof carboxymethylcellulose by the cellulase in the detergent, it isdesirable that carboxymethylcellulose is granulated or coated before theincorporation in the composition.

The detergent compositions of this invention may be incubated withcellulose containing fabric, for example soiled fabrics, in industrialand household uses at temperatures, reaction times and liquor ratiosconventionally employed in these environments. The incubationconditions, i.e., the conditions effective for treatingcellulose-containing fabrics with detergent compositions according tothe present invention, will be readily ascertainable by those of skillin the art. Accordingly, the appropriate conditions effective fortreatment with the present detergents will correspond to those usingsimilar detergent compositions that include known cellulases.

Detergents according to the present invention may additionally beformulated as a pre-wash in the appropriate solution at an intermediatepH where sufficient activity exists to provide desired improvementssoftening, depilling, pilling prevention, surface fiber removal orcleaning. When the detergent composition is a pre-soak (e.g., pre-washor pre-treatment) composition, either as a liquid, spray, gel or pastecomposition, the cellulase enzyme is generally employed from about0.0001 to about 1 weight percent based on the total weight of thepre-soak or pre-treatment composition. In such compositions, asurfactant may optionally be employed and when employed, is generallypresent at a concentration of from about 0.005 to about 20 weightpercent based on the total weight of the pre-soak. The remainder of thecomposition comprises conventional components used in the pre-soak,e.g., diluent, buffers, other enzymes (proteases), and the like at theirconventional concentrations.

It is contemplated that compositions comprising cellulase enzymesdescribed herein can be used in home use as a stand alone compositionsuitable for restoring color to faded fabrics (see, for example, U.S.Pat. No. 4,738,682, which is incorporated herein by reference in itsentirety) as well as used in a spot-remover and for depilling andantipilling (pilling prevention).

The use of the cellulase according to the invention may be particularlyeffective in feed additives and in the processing of pulp and paper.These additional industrial applications are described in, for example,PCT Publication No. 95/16360 and Finnish Granted Patent No. 87372,respectively.

In order to further illustrate the present invention and advantagesthereof, the following specific examples are given with theunderstanding that they are being offered to illustrate the presentinvention and should not be construed in any way as limiting its scope.

EXAMPLES Example 1 Temperature Stability Testing of EGIII and an EGIIIHomolog From Hypocrea schweinitzii

EGIII and an EGIII homolog derived from Hypocrea schweinitzii weretested to determine their stability under temperature stress. 0.3 mg/mlof enzyme was tested in 0.1 M MOPS, at pH 7.3, 48° C. and the activityon oNPC measured and compared over time. The experiment was run twotimes. The natural log of the activity was plotted against time ofincubation, and the rate constant for inactivation obtained from theslope of the straight line. Results for various mutants are provided inTable 1.

TABLE 1 Half Life of EGIII and a Homolog EGIII Homolog from HypocreaTrichoderma reesei EGIII schweinitzii 20.2 3.40 21.2 3.90

As shown in Table 1, the half-life of EGIII from T. reesei issignificantly greater than that of the EGIII homolog from Hypocreaschweinitzii.

Example 2 Wash Tests With EGIII and an EGIII Homolog From Hypocreaschweinitzii

EGIII was compared to a homologous enzyme derived from from Hypocreaschweinitzii. The amino acid sequence of the enzyme from Hypocreaschweinitzii is provided in FIG. 3 in alignment with the sequence ofEGIII. As shown in FIG. 3, the amino acid sequence of the two enzymeswas found to be identical except for the residues in bold correspondingto positions 7, 11, 16, 35, 39, 41, 63, 66, 77, 91, 143, 163, 167, and188.

In the wash test, three different enzyme mixtures (a) EGIII, (b) anEGIII homolog derived from Hypocrea schweinitzii, and (c) a combinationof the two enzymes were prepared and mixed separately with a standardLAS containing granular detergent (4 g/L) in water having a hardness of70 ppm CaCO₃ (2:1 Ca:Mg) at 40° C. in a Terg-o-Tometer with cottonswatches. The agitation was 125 rpm and the test was run for 2.5 hours.After the test, the swatches were removed from the Terg-o-Tometer, driedin a tumble drier and the level of pilling compared to a panel offabrics pilled to varying extents. The EGIII-like enzyme from Hypocreaschweinitzii showed no depilling performance at any concentration. Bycontrast, EGIII showed depilling performance that increased inaccordance with the enzyme concentration. The equivalent performance ofEGIII spiked into the Hypocrea schweinitzii broth containing theEGIII-like enzyme indicated that it was not a component of the brothwhich prevented performance of the EGIII-like enzyme but, instead, theenzyme itself which had poor stability and performance.

This experiment illustrated that stability of the EGIII-like enzyme fromHypocrea schweinitzii is far inferior to EGIII. In fact, the relatedenzyme exhibited no activity in the LAS containing detergent whereasEGIII retained excellent activity.

Example 3 Thermal Stability of EGIII Variants

Site-directed mutagenesis was performed to incorporate amino acidsubstitutions in T. reesei EGIII. The amino acids substituted into theEGIII were those at homologous locations in the H. Schweinitzii EGIIIhomolog.

PCR was performed according to well-known techniques.

TABLE 2 PCR primers Variant Forward primer Reverse Primer W7Y GCT GTGACC AGT ACG GTG AAG GTT GCG TAC CAA CCT TCA C TGG TCA CAG C T11S/T GGCAAC CTT CTC TGG GGT TGT TGC TGA CGA 16I CAA CGG CTA CAT CGT TGT AGC CGTTGC CAG CAG CAA CAA CC AGA AGG TTG CC A35S GGC TGC GTG ACG TCG GAG CGATAC CGA CGT GTA TCG CTC CAC GCA GCC S39N GGT ATC GCT CAA CGG GGA GGC CCCGCC GTT CGG GGC CTC C GAG CGA TAC C G41A CGC TCA GCG GCG CGG GCC AGG AGGCCG CGC CCT CCT GGC CGC TGA GCG S63V CGT ACC AGA ACG TTC GGA ATG GCA ATCTGA AGA TTG CCA TTC C ACG TTC TGG TAC G A66N CTC TCA GAT TAA CAT CCT CTTCTG GGG AAT TCC CCA GAA GAG G GTT AAT CTG AGA G S77G CGT CAA CAG CAT CGGGGG CAT GCT GCC GAT CAG CAT GCC C GCT GTT GAC G N91D GCG GGA GCG ACA TCCGCA ACA TTA GCG CGG GCG CTA ATG TTG C ATG TCG CTC CCG C S143T CGT CGGTGG CCA GAC GCG TCC AGG TCT GGC CTG GAC GC CAC CGA CG T163S CCT TTG TGGCCC AGA GGT AGT GTT GCT CTG GCA ACA CTA CC GGC CAC AAA GG N167S CCA ACACTA CCA GCT CAT CTC CGC TGT AGC ACA GCG GAG ATG TGG TAG TGT TGG A188GGGA TAC AAC GCT GGA CAT ATT GGC CTC CAG GGC CAA TAT G CGT TGT ATC C

Briefly, DNA that encodes T. reesei EGIII was amplified from a cDNAclone (Ward, et al., Proc. of the Tricel Symposium on “Trichodermareesei cellulases and other hydrolases.” Espoo, Finland 1993 Ed.Suominen, P. and Reinikanen, T. Foundation for Biotechnical andIndustrial Research. 8, pp 153-158; and U.S. Pat. No. 5,475,101) usingPCR primers that introduced a Bgl II restriction endonuclease site atthe 5′ end of the egl3 gene (immediately upstream of the first ATGcodon) and an Xba I site at the 3′ end (immediately downstream of the“stop” codon). The amplified fragment was then digested with Bgl II andXba I, and ligated into pUC19 digested with Bgl II and Xba I.

Variants were made in this plasmid using the QuikChange™ mutagenesismethods (Strategene). The variant genes were then subcloned into theAspergillus expression vector pPGPT-pyrG (Berka and Barnett, Biotech.Adv. 7:127 (1989)). Vectors carrying the variant genes were thentransformed into A. niger var awamori and the resultant strains grown inshake-flask cultures (WO 98/31821).

EG III variants were then purified from cell-free supernatants of thesecultures by column chromatography. Briefly, approximately 1 mL ofPharmacia Butyl Sepharose (Fast Flow) resin per 10 mg of EGIII wasloaded into a disposable drip column with 0.5 M. ammonium sulfate. Thecolumn was then equilibrated with 0.05 M Bis Tris Propane and 0.05 Mammonium acetate at pH 8.

The EGIII-like cellulase containing supernatants were treated overnightwith 0.18 mg/mL of endoglucanase H at 37° C. Ammonium sulfate was addedto the treated supernatants to a final concentration of approximately0.5 M. After centrifugation, the supernatant was loaded onto the column.The column was then washed with 3 volumes equilibration buffer and.theneluted with 2×1 volumes of 0.05 M Bis Tris Propane and 0.05 M ammoniumacetate, pH 8. Each volume of flow through was collected as a separatefraction with the EGIII-like cellulase appearing in the second fraction.

Equilibrium CD experiments were performed on an Aviv 62DS or 62ADSspectrophotometer, equipped with a 5 position thermoelectric cell holdersupplied by Aviv. Buffer conditions were 50 mM bis-tris propane and 50mM ammonium acetate adjusted to pH 8.0 with acetic acid. The finalprotein concentration for each experiment was in the range of 5-30 mM.Data was collected in a 0.1 cm path length cell.

Spectra were collected from 265-˜210 nm. Thermal denaturations wereperformed at 217 nm from 30 to 90° C. with data collected every twodegrees. The equilibration time at each temperature was 0.1 minutes anddata was collected for 4 seconds per sample.

The remainder of the pH 8.0 sample was divided into 5×400 μL aliquots.Two samples were adjusted to pH 5 and 7 with acetic acid and two otherswere adjusted to pH 9 and 10 with sodium hydroxide. Thermaldenaturations of all five samples were performed simultaneously asdescribed above. The melting points were determined according to themethods of Luo, et al., Biochemistry 34:10669 and Gloss, et al.,Biochemistry 36:5612.

TABLE 3 Thermal stability of EGIII variants Amino acid substitutions ΔT_(m) Tm (° C.) Fit error T. reesei EGIII 0.00 54.43 0.20 W7Y −1.0353.40 0.24 T11S/T16I 1.07 55.50 0.13 A35S −4.03 50.40 0.14 S39N 0.4754.90 0.17 G41A 2.47 56.90 0.11 S63V −0.83 53.60 0.11 A66N 0.07 54.500.10 S77G 0.07 54.50 0.09 N91D 0.47 54.90 0.17 S143T 0.47 54.90 0.12T163S 0.27 54.70 0.07 N167S 0.17 54.60 0.10 A188G 0.47 54.90 0.17

As can be seen from Table 3, most of the substitutions increased themelting point over that of wild type EGIII.

Example 4 Specific Activity of Variant EGIII Cellulases

To assay for specific activity, a NPC hydrolysis assay was used. In amicrotiter plate, 100 μl 50 mM sodium acetate, pH 5.5 and 20 μl 25 mg/mLo-NPC (o-Nitrophenyl o-D-Cellobioside (Sigma N 4764)) in assay bufferwas added. The plate was incubated for 10 minutes at 40° C.

Once equilibrated, 10 μL EGIII-like cellulase was added and the plateincubated at 40° C. for another 10 minutes. To quench the hydrolysis andstop the reaction, 70 μL of 0.2 M glycine, pH 10.0 was added. The platewas then read in a microtiter plate reader at 410 nm. As a guide, 10 μLof a 0.1 mg/ml solution of T. reesei EGIII provided an OD of around 0.3.

The concentration of EGIII-like cellulase was determined by absorbanceat 280 nm where the extinction coefficient was 78711 M⁻¹ cm⁻¹ or 3.352g/L⁻¹ experimentally determined by the method of Edelhoch as describedin Pace, et al., Pro. Sci. 4:2411 (1995).

TABLE 4 Specific Activity of Variant EGIII Cellulases Specific ActivityVariant (relative to WT EGIII) WT EGIII 1.00 W7Y 1.09 T11S/T16I 1.02A35S 0.79 S39N 0.82 G41A 0.90 S63V 0.68 A66N 1.00 S77G 1.02 N91D 0.89S143T 0.89 T163S 0.99 N167S 0.94 A188G 0.86

Surprisingly, the substitutions had little or no affect on the specificactivity of the variants compared to wild type EGIII.

4 1 232 PRT T. reesei 1 Met Lys Phe Leu Gln Val Leu Pro Ala Leu Ile ProAla Ala Leu Ala 1 5 10 15 Gln Thr Ser Cys Asp Gln Trp Ala Thr Phe ThrGly Asn Gly Tyr Thr 20 25 30 Val Ser Asn Asn Leu Trp Gly Ala Ser Ala GlySer Gly Phe Gly Cys 35 40 45 Val Thr Ala Val Ser Leu Ser Gly Gly Ala HisAla Asp Trp Gln Trp 50 55 60 Ser Gly Gly Gln Asn Asn Val Lys Ser Tyr GlnAsn Ser Gln Ile Ala 65 70 75 80 Ile Pro Gln Lys Arg Thr Val Asn Ser IleSer Ser Met Pro Thr Thr 85 90 95 Ala Ser Trp Ser Tyr Ser Gly Ser Asn IleArg Ala Asn Val Ala Tyr 100 105 110 Asp Leu Phe Thr Ala Ala Asn Pro AsnHis Val Thr Tyr Ser Gly Asp 115 120 125 Tyr Glu Leu Met Ile Trp Leu GlyLys Tyr Gly Asp Ile Gly Pro Ile 130 135 140 Gly Ser Ser Gln Gly Thr ValAsn Val Gly Gly Gln Ser Trp Thr Leu 145 150 155 160 Tyr Tyr Gly Tyr AsnGly Ala Met Gln Val Tyr Ser Phe Val Ala Gln 165 170 175 Thr Asn Thr ThrAsn Tyr Ser Gly Asp Val Lys Asn Phe Phe Asn Tyr 180 185 190 Leu Arg AspAsn Lys Gly Tyr Asn Ala Ala Gly Gln Tyr Val Leu Ser 195 200 205 Tyr GlnPhe Gly Thr Glu Pro Phe Thr Gly Ser Gly Thr Leu Asn Val 210 215 220 AlaSer Trp Thr Ala Ser Ile Asn 225 230 2 702 DNA T. longibrachiatum 2atgaagttcc ttcaagtcct ccctgccctc ataccggccg ccctggccca aaccagctgt 60gaccagtggg caaccttcac tggcaacggc tacacagtca gcaacaacct ttggggagca 120tcagccggct ctggatttgg ctgcgtgacg gcggtatcgc tcagcggcgg ggcctcctgg 180cacgcagact ggcagtggtc cggcggccag aacaacgtca agtcgtacca gaactctcag 240attgccattc cccagaagag gaccgtcaac agcatcagca gcatgcccac cactgccagc 300tggagctaca gcgggagcaa catccgcgct aatgttgcgt atgacttgtt caccgcagcc 360aacccgaatc atgtcacgta ctcgggagac tacgaactca tgatctggct tggcaaatac 420ggcgatattg ggccgattgg gtcctcacag ggaacagtca acgtcggtgg ccagagctgg 480acgctctact atggctacaa cggagccatg caagtctatt cctttgtggc ccagaccaac 540actaccaact acagcggaga tgtcaagaac ttcttcaatt atctccgaga caataaagga 600tacaacgctg caggccaata tgttcttagc taccaatttg gtaccgagcc cttcacgggc 660agtggaactc tgaacgtcgc atcctggacc gcatctatca ac 702 3 234 PRT T. reesei 3Met Lys Phe Leu Gln Val Leu Pro Ala Leu Ile Pro Ala Ala Leu Ala 1 5 1015 Gln Thr Ser Cys Asp Gln Trp Ala Thr Phe Thr Gly Asn Gly Tyr Thr 20 2530 Val Ser Asn Asn Leu Trp Gly Ala Ser Ala Gly Ser Gly Phe Gly Cys 35 4045 Val Thr Ala Val Ser Leu Ser Gly Gly Ala Ser Trp His Ala Asp Trp 50 5560 Gln Trp Ser Gly Gly Gln Asn Asn Val Lys Ser Tyr Gln Asn Ser Gln 65 7075 80 Ile Ala Ile Pro Gln Lys Arg Thr Val Asn Ser Ile Ser Ser Met Pro 8590 95 Thr Thr Ala Ser Trp Ser Tyr Ser Gly Ser Asn Ile Arg Ala Asn Val100 105 110 Ala Tyr Asp Leu Phe Thr Ala Ala Asn Pro Asn His Val Thr TyrSer 115 120 125 Gly Asp Tyr Glu Leu Met Ile Trp Leu Gly Lys Tyr Gly AspIle Gly 130 135 140 Pro Ile Gly Ser Ser Gln Gly Thr Val Asn Val Gly GlyGln Ser Trp 145 150 155 160 Thr Leu Tyr Tyr Gly Tyr Asn Gly Ala Met GlnVal Tyr Ser Phe Val 165 170 175 Ala Gln Thr Asn Thr Thr Asn Tyr Ser GlyAsp Val Lys Asn Phe Phe 180 185 190 Asn Tyr Leu Arg Asp Asn Lys Gly TyrAsn Ala Ala Gly Gln Tyr Val 195 200 205 Leu Ser Tyr Gln Phe Gly Thr GluPro Phe Thr Gly Ser Gly Thr Leu 210 215 220 Asn Val Ala Ser Trp Thr AlaSer Ile Asn 225 230 4 234 PRT H. schweinitzii 4 Met Lys Phe Leu Gln ValLeu Pro Ala Ile Leu Pro Ala Ala Leu Ala 1 5 10 15 Gln Thr Ser Cys AspGln Tyr Ala Thr Phe Ser Gly Asn Gly Tyr Ile 20 25 30 Val Ser Asn Asn LeuTrp Gly Ala Ser Ala Gly Ser Gly Phe Gly Cys 35 40 45 Val Thr Ser Val SerLeu Asn Gly Ala Ala Ser Trp His Ala Asp Trp 50 55 60 Gln Trp Ser Gly GlyGln Asn Asn Val Lys Ser Tyr Gln Asn Val Gln 65 70 75 80 Ile Asn Ile ProGln Lys Arg Thr Val Asn Ser Ile Gly Ser Met Pro 85 90 95 Thr Thr Ala SerTrp Ser Tyr Ser Gly Ser Asp Ile Arg Ala Asn Val 100 105 110 Ala Tyr AspLeu Phe Thr Ala Ala Asn Pro Asn His Val Thr Tyr Ser 115 120 125 Gly AspTyr Glu Leu Met Ile Trp Leu Gly Lys Tyr Gly Asp Ile Gly 130 135 140 ProIle Gly Ser Ser Gln Gly Thr Val Asn Val Gly Gly Gln Thr Trp 145 150 155160 Thr Leu Tyr Tyr Gly Tyr Asn Gly Ala Met Gln Val Tyr Ser Phe Val 165170 175 Ala Gln Ser Asn Thr Thr Ser Tyr Ser Gly Asp Val Lys Asn Phe Phe180 185 190 Asn Tyr Leu Arg Asp Asn Lys Gly Tyr Asn Ala Gly Gly Gln TyrVal 195 200 205 Leu Ser Tyr Gln Phe Gly Thr Glu Pro Phe Thr Gly Ser GlyThr Leu 210 215 220 Asn Val Ala Ser Trp Thr Ala Ser Ile Asn 225 230

We claim:
 1. A feed additive comprising a variant EGIII cellulasecomprising a substitution at a residue which is sensitive to surfactantand/or temperature stress, wherein said variant EGIII cellulase isderived from T. reesei EGIII cellulase.